The interaction between cancer cells and the pancreatic stroma has been implicated in pancreatic cancer progression and resistance to chemotherapy and radiation. Based on our preliminary results demonstrating prominent tissue transglutaminase (TG2) expression and activity in the pancreatic tumor millieu, we hypothesized that TG2 secreted by pancreatic cancer cells modulates stroma, promotes cancer cell survival, and impairs chemotherapy delivery. We propose to test the hypothesis by three specific aims: Aim 1: Measure proliferation and activation of pancreatic stromal cells in response to TG2 secreted by pancreatic cancer cells. Aim 2: Demonstrate that TG2 knockdown or its enzymatic inhibition regulate stroma composition in pancreatic tumors. Aim 3: Demonstrate that TG2 knockdown or its enzymatic inhibition increases gemcitabine delivery into the pancreatic milieu. Aim 1 will use co-culture experiments to measure the effects of secreted TG2 on proliferation, survival and activation of fibroblasts and endothelial cells. Cytokines and growth factors secreted by fibroblasts will be measured in media conditioned by stromal and cancer cells by using Multiplex ELISA. Aims 2 and 3 will use orthotopic pancreatic xenografts generated from pancreatic cancer cells in which TG2 was knocked down by using stable transfection of sh-RNA or antisense construct targeting TG2, or TG2 will be inhibited by using KCC009, a specific enzymatic inhibitor. Endpoints of the experiments are measurement and comparison of: fibrosis (immunostaining for collagen and trichrome stain), number and proliferation of stromal cells (myofibroblasts and pancreatic stellate cells), angiogenesis (microvessel density), survival and proliferation of pancreatic cancer cells, and penetrance of chemotherapy into pancreatic tissue. For the latter endpoint, we will measure the concentration of gemcitabine within pancreatic tissue derived from cancer cells expressing normal or low TG2 levels by using HPLC mass spectrometry. In summary, successful demonstration of the role of tissue transglutaminase in modulation of the pancreatic stroma will provide a strong rationale for investigating further TG2 inhibition as an anti-cancer strategy or as means to sensitize pancreatic tumors to chemotherapy. These studies have direct clinical relevance to pancreatic cancer patients.